As the regulations on automobile exhaust emissions have been strict, higher level analyses or evaluations of fuels are required more than they used to be. For example, microanalysis dealing with trace amounts of compounds such as polynuclear aromatics are required for light oil.
On the other hand, extremely small amount of samples or the samples including low concentration compounds have been routinely analyzed in the field of environmental science. The demand for the analysis with high sensitivity or high selectively also have increased in the other analytical field.
When analyzing these samples, their concentration is important. And, the analytical results depend on the quality of the procedure for concentration. In general, the method of vaporizing the solvent at room temperature or a heated temperature under an atmospheric pressure or a reduced pressure has been used for concentration of the solution samples. For example, an evaporator has been used as a typical concentration method. In addition, the mini-column packed with the packing material for liquid chromatography and so on has recently been used to extract the solute.
In the treatment of low concentration samples, the following problem is likely to arise: the concentration under an atmospheric or reduced pressure loses the solutes where boiling points are close to that of the solvent, and the concentration using the mini-column will dilute the solute to be obtained with a large amount of solvent or makes the solute go away in vapor at recovery.
By the way, automation of the analytical procedures has been promoted and the apparatuses for them have been developed.
The analysis of complex mixture sample is generally classified into pre-treatment, separation, identification and quantitation. The automation of these procedures serves to not only speed-up and labor-saving of the procedures but also improvement of accuracy, sensitivity and reproducibility of analytical results.
Among the above procedures, the method for pre-treatment is varied, depending on the sample conditions or the purposes of analysis. Accordingly, the effect of the automation is little except the case where the similar samples are treated repeatedly. On the other hand, the automation of the procedures in separation, identification and quantitation will be widely used. A typical example of the above automation is GC/MS (gas chromatography/mass spectrometer) in which a gas chromatograph is combined with a mass spectrometer. MS, which is effective in identification and structural analysis of very small amount of organic compounds, has been widely used in the analytical field, since it was combined with GC in 1964. Nowadays, the GC/MS is popularly used as a most powerful analytical systems. However, GC/MS can analyze only the samples which evaporate in GC column (where the temperature is lower than 350.degree. C. and the pressure is lower than 150 kPa). In other words, GC/MS is not applicable to the samples with high boiling point which cannot be separated by this column. As a method for solving the problem, the following method and the like, is employed in which the high boiling point substances are thermally cracked and introduced into GC/MS as low boiling-point substances. However, HPLC, GPC, SFC and so on has generally been used as the separation method for such samples.
The number of the kinds of organic compounds subjected to HPLC is much larger than that subjected to GC, as the number of the kinds of organic compounds exponentially increases with molecular weight.
From the above background, the methods for combining an analytical instrument for identification and structural analysis, such as MS, IR and NMR, with a separation apparatus, such as HPLC, GPC or SFC, have been developed so far.
The most difficult problem in combining HPLC with MS, IR or NMR had been to treat the solvent from HPLC, because the weight of the solvent corresponds to 100 to 1,000 times as much as the carrier gas from GC. It was believed at first that it had been more difficult to combine HPLC with MS than with IR or NMR, because MS must be maintained at a high vacuum. However, contrary to the above prediction , many interfaces for LC/MS were developed. As a result, most LC/MS are more advanced systems than LC/IR and LC/NMR. In most LC/MS interfaces, the solvent from HPLC is used effectively as an ionization-assisting agent.
On the other hand, combining HPLC with NMR, in which a sample is measured in the state of solution, was thought to be easier than with MS. However, the development of the interfaces for LC/NMR were delayed compared with that of LC/MS. LC/NMR systems marketed at present are the same in principle as the system developed first in 1978. Namely, these LC/NMR systems accept HPLC effluent without concentration. In the system, the NMR spectrum of the sample is subtracted with the spectrum of the solvent, which was measured in advance, by the data processing system.
Therefore, the problem that the peaks of solute disappear with the peaks of solvent is likely to arise, when the concentration of the sample is low. The NMR has the lowest sensitivity among the above three analytical instruments. In addition, it has disadvantage that the sensitivity of carbon nuclei (.sup.13 C) which provide important information for the analysis of organic compound, is much lower than that of proton nuclei (.sup.1 H). Accordingly, even in LC/NMR which is commercially available now, the On-Flow measurement is limited to the .sup.1 H-NMR measurement of high-concentration sample.
The development of LC/IR has been further behind LC/NMR. In infrared spectrometer (4,000 to 400 cm.sup.-1), HPLC solvents such as n-hexane, methylenechloride, chloroform, tetrachloroethane exhibit absorbance at the wave number which is important for analyzing organic compounds. Consequently, the subtraction of the solvent's peaks by data processing system is more difficult in LC/NMR systems. Namely, the solvent must be completely removed by hardware to make LC/IR a practical system
Namely, the most serious problem under the development of LC/IR systems and LC/NMR systems were how to eliminate the solvent in the effluent from HPLC.
When we concentrate the effluents from the HPLC, we will be confronted with the following problems.
The first problem arises when the effluents contain the substance where boiling point is low or slightly higher than the solvent. Even if we mildly concentrate such solution under an atmospheric pressure at room temperature, the substance where the boiling point is close to that of the solvent will vaporize with the solvent.
The second problem arises when the effluent from the HPLC is concentrated completely. After complete concentration, the solute becomes high in viscosity and loses its fluidity. In this case, the transfer of the concentrated solute to the subsequent analytical instrument during the concentration becomes difficult. The degree of influence of the decrease in fluidity with concentration differs between LC/NMR and LC/IR.
There is no need to concentrate completely the solution from HPLC as the NMR measures the sample in the state of solution. The HPLC effluent needs to be concentrated up to the level which depends on the sensitivity of NMR. The sensitivity depends on the kind of target nuclei and the measurement mode. That is, NMR is required to continuously transfer enriched effluent to NMR probe, while maintaining a predetermined concentration rate.
On the other hand, on LC/IR, the solvent which is transferred from HPLC needs to be removed completely, because the solvent mostly affects to the IR spectrum.
The GC has been used popularly as an instrument for separating a complex mixture which consists of the substances with the boiling point lower than 350.degree. C. In the GC, substances injected into the column are carried by the carrier gas while they travel reciprocately between the liquid film coated on the inner surface of the column or on the surface of the packing material in the packed column and the carrier gas which flows through the column. The moving rate of the substance depends on its boiling point and its polarity, and gas chromatographic conditions such as the compositions of the liquid film, the temperature of the column and the flow rate of carrier gas. For example, the lower the boiling point of the substance is, or the lower the affinity of the substance to the liquid film is, the faster the substance will be eluted.
As described above, GC is a separation method which has extremely high separating power, which is enough to separate the mixture which consists of the components where the boiling point, polarity or optical activity are slightly different from each other. At the beginning when GC when first developed, the column packed with chemical coated clay or the column packed with polymer had been used as the column. The capillary column, the inside of which is coated and crosslinked with chemicals has generally been used as of late.
Though GC is a method where the separation power is very high, the amount of liquid sample which can be injected is limited to about 10 .mu.L. Accordingly, the amount of the solute which can be recovered after concentration of thin solution less than 1% will be about 0.1 .mu.L in total. If the solute recovered is a complex mixture, the analytical instrument which is able to clear the composition is very limited.
About 1,000 times as much as the sample usually injected for GC (e.g., about 10 mL) must be separated and collected in order to clear the above mixture using the several marketed analytical instruments.
On the other hand, when two kinds of liquid chromatography where the separation mechanisms are different, for instance, the first liquid chromatography (e.g., normal phase HPLC) and the second liquid chromatography (e.g., reverse phase HPLC), are used as a series of chromatography, the performance of the second column in separation will be down, because the solvent from the first column enters into the second column and changes its conditions. Therefore, the second column must be conditioned with a lot of the solvent for the second chromatography in order to recover the original performance of the second column. In addition, in a method where an adsorbed substance is recovered by using back flush, when the back-flushed eluted substance is injected into the next column as it is, since the eluted substance is introduced into the next column together with a large amount of a solvent, there arises an elution layer of a broader band so that a drawback occurs to deteriorate the separating capacity.
The present invention has been developed in view of the aforementioned circumstances. It is therefore an object of the present invention to provide a method which can concentrate a solution containing a solute having a boiling point being close to a boiling point of a solvent, or a solution containing a solute having a polarity being different from a polarity of a solvent, without a loss of the solute so that the solution can be analyzed, and an apparatus for the same; and an apparatus which can make the concentrate obtained by the present method easily connectable with analyzing means to be applied later.